Individuals with type 2 diabetes exhibit excessive postprandial hyperglycemia, in part, due to a failure of elevated glucose and insulin to stimulate hepatic glucose uptake via an activation of glucokinase (GK). GK activity in the liver is regulated by changes in the amount of enzyme, its cellular location, and whether it is bound to an inhibitor (i.e. its regulatory protein, GKRP). In liver there exists a "glucose effect"; whereby, glucose per se is a major stimulant of its own utilization and/or storage as glycogen. Failure of elevated glucose to signal glucose uptake in the diabetic liver may arise from a defect in the dissociation of GK from GKRP and/or the translocation of GK from the nucleus to the cytoplasm. We observed in a model of obesetype 2 diabetes, Zucker diabetic fatty (ZDF) rats, that chronic hyperglycemia resulted in the loss of the "glucose effect". This glucose toxicity prevented the activation and dissociation of GK from GKRP and was reversed after administering an agent that normalized hyperglycemia. Preventing hyperglycemia allowed the diabetic liver to "sense" a rise in glucose and respond normally, yet the underlying mechanisms of hepatic glucose toxicity and of glucose signaling is not known. Therefore, we propose four aims: 1) Examine the effects of altering GK distribution on glucose utilization and glycogen synthesis by decreasing GKRP expression and/or increasing expression of cytosolic GK binding proteins. 2) Determine if a loss of a permissive effect of insulin and/or an increase in glucagon antagonism contributes to hepatic glucose toxicity. 3) Determine whether an inhibitory feedback signal from a pathway of glucose metabolism is involved. 4) Determine the involvement of systemic influences, such as autonomic nervous dysfunction, increased cytokines, or excessive release of insulin. To execute these aims we will use isotopic and chronic catheterization techniques to perform glucose and pancreatic clamps in conscious ZDF rats. The intracellular localization of enzymes will be assessed immunohistochemically by confocal microscopy. Chronic Hyperglycemia will be altered by a phlorizin analogue. The expression of some enzymes and proteins will be altered by gene or siRNA transfer using adenovirus vectors. We believe that experiments directed towards understanding the mechanism by which chronic hyperglycemia impairs the actions of glucose on the liver will provide the basis for improved management and therapeutic treatment of type 2 diabetes.